Copper enzymes perform diverse reactions that include O2 activation for the monooxygenation, dioxygenation and oxidation of substrates in natural product biosynthesis (related to a number of disease states), the 4 electron reduction of O2 to H2O in iron and copper metabolism and proton pumping for ATP synthesis, and the 2 electron reduction of N2O in denitrification. The active sites in these enzymes have different geometric and electronic structures: antiferromagnetically coupled binuclear copper sites, non-coupled binuclear copper sites, trinuclear copper clusters, heme-copper sites, a tetranuclear copper cluster, and mononuclear copper centers that perform multi-electron reactions in cofactor biogenesis. These active sites and their intermediates exhibit unique spectral features, which reflect their different electronic structures that direct specific reaction coordinates for catalysis. The broad goals of this research program are to use a wide range of spectroscopies (including the development of new methods), coupled to electronic structure calculations, enzymology, and model studies to define the molecular mechanism for each class of copper enzymes and to understand how the differences in active site geometric and electronic structures over these classes determine their diverse specific functions: a) 2 electron activation of O2 for electrophilic aromatic attack in the coupled binuclear copper enzymes; b) 1 electron O2 activation for H atom abstraction by the non-coupled binuclear copper enzymes; c) O2 activation for H-atom abstraction from the strong C-H bond in methane by the recently defined binuclear copper center in particulate methane monooxygenase; d) the coupling of the four one-electron oxidations of substrate to the 4 electron reduction of O2 to H2O by the trinuclear copper cluster containing multicopper oxidases; e) the coupling of the 4 electron reduction of O2 to H2O in the heme-copper oxidases to proton pumping; f) N2O activation by the tetranuclear copper cluster site; g) the activation of O2 or substrate by a reduced or oxidized, respectively, mononuclear copper center. These studies provide molecular level insight into reactivity and define structure/function correlations over the O2 and N2O activating copper enzymes. They are of fundamental importance towards understanding pathogenesis and provide structural and mechanistic insight for drug design, medical device development, and the generation of new catalysts. PUBLIC HEALTH RELEVANCE: Copper proteins play critical roles in Fe, Cu and O2 metabolism, are directly related to a range of genetic diseases (oculocutaneous albinism1, aceruloplasminemia,2 etc.) and health issues (Alzheimer's,3 atherosclerosis,4 control of neurotransmiters,5,6 etc.) and are important in natural product biosynthesis,7,8 biotechnology9 (oxidoreductases,10 biofuel cells11,12), detoxification,13 and the bioremediation of greenhouse gases (N2O,14 CH415,16). Understanding Cu biochemistry on a molecular level provides mechanisms to improve or inhibit these processes, and enable drug design and the development of implantable devices.17